Minimal-invasiv teknikk for Injeksjon i Rat Optic Nerve
Summary
Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.
Abstract
The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.
Introduction
Synsnerven gir en ideell beliggenhet for sentralnervesystemet (CNS) regenerativ forskning inkludert oftalmologiske forhold som optikusnevritt, glaukom og traumer. Injeksjoner av en rekke stamceller enten har vist effekt eller vist lovende i å erstatte tapt myelin, øke aksonal teller og / eller forebygge degenerative sykdommer. 1,2
Den menneskelige synsnerven inneholder ca 1,2 millioner parallelle aksoner reiser fra netthinnen til chiasm med en diameter på ca 3,0-3,5 mm. 3 For å modellere menneskelige sykdommer i laboratoriet, har rotta vært brukt ofte. Den voksne rotteoptisk nerve inneholder omtrent 100.000 aksoner innenfor en diameter på ca. 0,5 mm. 4 En av de store begrensningene i CNS regenerative forskning er direkte benfrie tilgang. Komplikasjoner og kirurgisk risiko for dyret er høyere når hodeskallen eller vertebrae er fjernet. I likhet med fordeleneminimal-invasive tilnærminger i ryggraden, 5 direkte synsnerven injeksjoner uten å åpne skallen tilby reduserte komplikasjoner og en raskere bedring.
Denne teknikken har blitt anvendt i tidligere studier. 6 I dette manuskriptet og tilhørende video demonstrerer vi en minimalt invasiv prosedyre for å injisere stamceller i rotte synsnerven.
Protocol
Representative Results
Discussion
Direct injection into the optic nerve of stem cells or other products intended to facilitate regeneration provides a convenient model compared to other means of injections into the CNS. This technique takes less time, requires less total anesthesia, avoids drilling or removing skull or bone tissue, reduces complications rates and allows for more rapid recovery following surgery.
The most critical steps in this protocol include: 1. Adequate hemostasis in the surgical field to allow clear visua…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This study was supported by NeuralStem, Inc., and Johns Hopkins Project RESTORE.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Lewis rat | Charles River | 4 | Any rat strain will work. |
| Anesthesia machine | Surgivet | CDS9000 | CDS 9000 Small Animal Anesthesia Machine – Pole Mount |
| Infusion pump | Stoelting | 53129 | |
| Dissection microscope | National Optical | 409-411-1105 | |
| Fiber-optic light source | Fisher Scientific | 12-562-21 | |
| Dissection and Stereotaxic Instrument | Stoelting | 51400 | |
| Pipette Puller | Kopf | 750 | |
| Pipettes | World Precision Instruments | 18150-6 | |
| Disposable scalpel blades | Harvard Apparatus | 810-15-021 | |
| Iridectomy scissors | Electron Microscopy Sciences | Uniband LA-4XF |
Riferimenti
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